Monitoring Binding Kinetics with Ultrasonic Spectroscopy

Binding of Mg2+ to polyriboadenilic acid, poly (A)

Binding of cytidine 2'-monophosphate (2'-CMP) to ribonuclease A (RNase A)

Hydrolysis of maltoheptaose by α-amylase

Monitoring of immunochemical reactions

Hydrolysis of Maltodextrin by α-amylase

 

 

Application 1: Binding of Mg2+ to polyriboadenilic acid, poly (A)

A concentrated solution of MgCl2 was added stepwise into the measuring ultrasonic cell containing 1 ml of aqueous solution of poly(A) (analogue of well known RNA) and into the reference ultrasonic cell containing 1 ml of buffer. The device measured the difference in ultrasonic parameters of the samples in the measuring and the reference cells, thus subtracting the contribution of MgCl2. Therefore the plotted changes of ultrasonic velocity and attenuation represent the interaction of magnesium ions with poly(A) only.  Binding of magnesium with poly(A) results in the initial decrease of ultrasonic velocity caused by the release of hydration water from the coordination shell of Mg2+ ions and atomic groups of the polymer. The compressibility of water in the hydration shells of the ligand and the polymer is less than the compressibility of the bulk water, therefore transferring of hydration water into the bulk water increases the total compressibility of the solution, thus reducing the ultrasonic velocity. When all available sites on the polymer are occupied by the ligand the curve levels off. The total drop in ultrasonic velocity is linked with the number of water molecules excluded from the coordination shell of Mg2+ allowing to make structural characterisation of the complex. Binding constants and stoichiometries can be calculated from the shape of the curve. At high concentrations of magnesium electrostatically neutralised polymer molecules begin to form aggregates. The scattering of ultrasonic waves by the aggregates leads to the increase in attenuation. Additional dehydration and intrinsic compressibility of the aggregates results in the decrease of ultrasonic velocity at this stage. It is important that all these measurements do not require any optical activity of the ligand and the polymer and optical transparency of the medium.  As the hydration, or solvation, effects are involved in most of reactions in solutions the high-resolution ultrasonic spectroscopy has a potential of being a universal technique for their analysis.

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Application 2: Binding of cytidine 2'-monophosphate (2'-CMP) to ribonuclease A (RNase A)

2'-CMP inhibits RNase A activity, being isomeric with the natural RNA structure. The binding site for 2'-CMP lies deep within the cleft of the enzyme.

A concentrated solution of 2'-CMP was added stepwise into the measuring ultrasonic cell containing 1 ml of aqueous solution of protein and into the reference ultrasonic cell containing 1 ml of buffer. The device (HR-US 102) measured the difference in ultrasonic parameters of the samples in the measuring and the reference cells, thus subtracting the contribution of 2'-CMP. Therefore the plotted changes of ultrasonic velocity represent the interaction of the ligand with protein only.  Binding of 2'-CMP to enzyme results in the initial decrease of ultrasonic velocity caused by the release of hydration water from the coordination shell of ligand and atomic groups of the polymer. The compressibility of water in the hydration shells of the ligand and the polymer is less than the compressibility of bulk water, therefore transferring of hydration water into the bulk water increases the total compressibility of the solution, thus reducing the ultrasonic velocity. When all available sites on the polymer are occupied by the ligand the curve levels off.

The total drop in ultrasonic velocity is linked with the number of water molecules excluded from the coordination shell within binding site allowing to make structural characterisation of the complex. Binding constants and stoichiometry can be calculated from the shape of the curve.

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Application 3: Hydrolysis of maltoheptaose by α-amylase

A further example of enzyme activity is a study of the hydrolysis of maltoheptaose by α-amylase. A 5g sample of enzyme was added to a 3.5 mM aqueous solution of the sugar, and the resulting reaction was followed by the continuous monitoring of changes in ultrasonic velocity. As the reaction proceeds, ultrasonic velocity increases because the hydration level of the product is higher than that of the starting substrate, as shown in the figure above. It is simple to recalculate the ultrasonic curve to give the time dependence of the amount of substrate that has been hydrolysed, providing the kinetic profile of the reaction, and allowing the enzyme's activity to be calculated.

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Application 4: Monitoring of immunochemical reactions

In the above figure, 1l doses of a commercially-available solution of an antibody specific to β-galactosidase were added stepwise to an ultrasonic cell containing 1 ml of a very dilute solution of the enzyme, and also into a reference cell containing 1ml of buffer. The HR-US 102 measured the difference in ultrasonic parameters between the two samples as the antibody was added, so the resulting plot is of the interaction between the antigen and the antibody only. Several different stages of antigen-antibody binding can be observed from the graph, corresponding to different antigen-antibody complexes. At a low antibody-enzyme ratio, in stage I, the antibody binds to all the available binding centres of the tetrameric enzyme, leading to a decrease in the ultrasonic velocity as the proteins' elasticity increases and water of hydration is released from the binding site. In stage II, as the concentration of antibody rises, the antigen-antibody links redistribute to achieve maximum intramolecular contacts. In the third stage, further addition of antibody results in the antigen-antibody complexes aggregating. This stage is characterised by the slow kinetics of the structural organisation of these aggregates, reflected in the fact that it takes around an hour for the ultrasonic parameters to reach a steady value. This is in sharp contrast to the first two stages, where the changes occur more quickly, and are essentially dependent on the efficiency of the mixing process. The total drop in ultrasonic velocity observed at each stage enables structural characterisation of the complex to be made, and both binding constants and stoichiometry can be calculated from the shape of the curve.

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Application 5: Hydrolysis of Maltodextrin by α-amylase

An important industrial enzyme α-amylase hydrolyses starch, glycogen, and related polysaccharides by randomly cleaving internal α-1,4-glucosidic linkages. It is used, for example, as an additive in detergents, for removal of starch sizing from textiles and proper formation of dextrin in baking. The figure above illustrates the ultrasonic (HR-US 102) monitoring of enzyme activity of α-amylase at 25C through the measurements of changes in ultrasonic velocity during the course of a hydrolysis of 10.4 mg/ml solution (0.02 M phosphate buffer, pH 6.9) by the enzyme.

Five microlitres of a 2 mg/ml amylase solution was added to a 1ml ultrasonic cell filled with maltodextrin solution in 0.02 M phosphate buffer at ten minutes of run. The hydrolysis of the maltodextrin by amylase causes an increase in ultrasonic velocity, because the hydration level of the product is higher than that of the maltodextrin substrate. The ultrasonic velocity curve was recalculated into the time dependence of the amount of substrate hydrolysed, that is, to the kinetic profile of the reaction. The enzyme activity calculated from this curve is 90 units/mg amylase (1 unit is defined as the amount of enzyme activity which liberate 1.0 mg of maltose in 1 min at 25oC).  Ultrasonic attenuation decreases during the reaction (insert in the figure above shows data at 14 MHz) as a result of a decrease in molecular weight of the substrate.  This decrease in the length of the substrate reduces the high frequency viscosity of the solution and therefore the attenuation of ultrasound.  This provides independent information on the change of molecular weight of the substrate in this reaction.

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